Research: Staphylococcus aureus is the most common cause of cutaneous and systemic infections in both healthy individuals as well as in patients with AIDS. The skin and anterior nares are commonly colonized by S. aureus, which can be the source of life-threatening complications. However, little is understood regarding the mechanisms that this organism employs in the disease processes. The ability of bacteria to adhere to, invade into, and grow in or on mammalian cells are important initial steps in infection. This proposal addresses each of these steps by infecting primary human keratinocytes and artificial skin systems with S. aureus. Both dead deepidermized dermis and a collagen- fibroblast matrix will be utilized as dermal substrates for keratinocyte growth and differentiation. Plate assays will be utilized to determine the ability of the bacteria to survive and proliferate in the tissue culture systems. Morphologic studies using light microscopy, immunofluorescence microscopy, and electron microscopy will be utilized to determine the localization of the bacteria and the interactions with undifferentiated and differentiated keratinocytes, as well as other cellular and extracellular matrix components. To identify S. aureus virulence genes, both positive (differential fluorescence induction using the green fluorescence protein) and negative (signature-tagged mutagenesis) selection techniques will be employed.